bioaffinity Authors
Yoichi Kumada, Reina Tanibata, Kimitaka Yamamoto, Hiroshi Noguchi, Alessandro Angelini, Jun-ichi Horiuchi
First author
Yoichi Kumada
Corresponding author
Yoichi Kumada
Publication Style
Journal name Journal of Immmunological Methods
Year
Volume, issue, pages
vol. 520, 113522
Abstract
In this study, we developed and demonstrated a latex turbidimetric immunoassay (LTIA) using latex beads immobilized with rabbit monoclonal single-chain variable fragments (scFvs) selected from an scFv-displayed phage library. Sixty-five different anti-c-reactive protein (anti-CRP) scFv clones were identified after biopanning selection using antigen-coupled multi-lamellar vesicles. By ranking antigen-binding clones using the apparent dissociation rate constant (appkoff) as a sorting index, scFv clones with a dissociation constant (KD free) ranging from 4.07 × 10−9 M to 1.21 × 10−11 M were isolated. Among them, three candidates (R2–6, R2–45, and R3–2) were produced in the culture supernatant at concentrations of 50 mg/L or higher in flask culture and maintained at considerably high antigen-binding activity in immobilized state on the CM5 sensor chip surface.
All the scFv-immobilized latexes (scFv-Ltxs) prepared were well-dispersed in 50 mM MOPS at pH 7.0, without additives for dispersion, and their antigen-dependent aggregation was sufficiently detectable. The reactivity of scFv-Ltx to antigen differed among the scFv clones, in particular, R2–45 scFv-Ltx detected the CRP with the highest signal. Furthermore, the reactivity of scFv-Ltx varied significantly with salt concentration, scFv immobilization density, and the type of blocking protein. Particularly, antigen-dependent latex aggregation improved significantly in all rabbit scFv clones when scFv-Ltx was blocked with horse muscle myoglobin compared with conventional bovine serum albumin; while their baseline signals in the absence of antigen were fully stable. Under optimal conditions, R2–45 scFv-Ltx exhibited greater aggregation signals with antigen concentrations higher than those produced by conventional polyclonal antibody-immobilized latex for CRP detection in LTIA. The methodology for rabbit scFv isolation, immobilization, and antigen-dependent latex aggregation demonstrated in the present study can be applicable to scFv-based LTIA for various target antigens.